The proposed research is an integrated genetic and biochemical attack on the problems of the identification, location and organization of genes on the mitochondrial genome of bakers' yeast, Saccharomyces cerevisiae. Major attention will be devoted to the analysis of the structure of the varl gene, discovered in the previous project period, and the segments flanking it. Taht gene and allele-specific insertions within it will be sequenced by the Maxam-Gilbert method using mitochondrial DNA from appropriate petite mutants. Some effort will be devoted to the development of improved sequencing procedures and to the cloning of mitochondrial DNA sequences in E. coli or yeast. The mechanism of gene conversion of insertions within the varl gene will be probed by genetic and sequencing experiments. Emphasis will also be given to the identification of additional structural genes on yeast mitochondrial DNA using the zygotic gene rescue protocol to achieve amplification of genes retained on the mitochondrial DNA of petite mutants. Each new gene product identified in that way will be assigned to a segment of the mitochondrial genome using methods developed by us for the study of the varl gene. Respiration deficient mutants will be isolated and analysed phenotypically in order to assign a function to each gene. As adjacent genes are identified, we will be able to test the Bernardi model of the organization of the yeast mitochondrial genome by obtaining the complete nucleotide sequence of at least two genes, one intragenic "spacer" and one regulatory (promoter) sequence.